The present invention relates, in general, to a method of modulating physiological and pathological processes and, in particular, to a method of modulating intra- and extracellular levels of oxidants such as superoxide radicals and hydrogen peroxide and thereby processes in which such radicals are a participant. The invention also relates to compounds and compositions suitable for use in such methods.
Oxidants are produced as part of the normal metabolism of all cells but also are an important component of the pathogenesis of many disease processes. Reactive oxygen species, for example, are critical elements of the pathogenesis of diseases of the lung, the central nervous system and skeletal muscle. Oxygen free radicals also play a role in modulating the effects of nitric oxide (NOxe2x80xa2). In this context, they contribute to the pathogenesis of vascular disorders, inflammatory diseases and the aging process.
A critical balance of defensive enzymes against oxidants is required to maintain normal cell and organ function. Superoxide dismutases (SODs), a family of metalloenzymes which catalyze the intra- and extracellular conversion of O2xe2x88x92 into H2O2 plus O2, and represent the first line of defense against the detrimental effects of superoxide radicals. Mammals produce three distinct SODs. One is a dimeric copper- and zinc-containing enzyme (CuZn SOD) found in the cytosol of all cells. A second is a tetrameric manganese-containing SOD (Mn SOD) found within mitochondria, and the third is a tetrameric, glycosylated, copper- and zinc-containing enzyme (EC-SOD) found in the extracellular fluids and bound to the extracellular matrix. Several other important antioxidant enzymes are known to exist within cells, including catalase and glutathione peroxidase. While extracellular fluids and the extracellular matrix contain only small amounts of these enzymes, other extracellular antioxidants are known to exist, including radical scavengers and inhibitors of lipid peroxidation, such as ascorbic acid, uric acid, and xcex1-tocopherol (Halliwell et al, Arch. Biochem. Biophys. 280:1 (1990)). The relative lack of extracellular antioxidant enzymes may reflect the possible function of extracellular reactive oxygen species as bioeffector molecules (Halliwell et al, Arch. Biochem. Biophys. 280:1 (1990)). The relative deficiency of such enzymes may also result in greater susceptibility to extracellular oxidant stresses.
The enzyme EC-SOD, in many extracellular locations, exists only at low concentrations. While its physiologic role in vivo is yet to be defined, in many extracellular locations, EC-SOD is not thought to function as a bulk scavenger of O2xe2x88x92. As indicated above, EC-SOD is a tetrameric Cu/Zn-containing glycoprotein with a subunit molecular weight of 30,000 (Marklund, Proc. Natl. Acad. Sci. USA 79:7634 (1982); Tibell et al, Proc. Natl. Acad. Sci. USA 84:6634 (1987); see also U.S. Pat. No. 5,130,245 and WO 91/04315). Biochemical data suggest that EC-SOD binds to heparan sulfate proteoglycans on endothelial cells, where it has been speculated to serve as a xe2x80x9cprotective coatxe2x80x9d (Marklund, J. Clin. Invest. 74:1398 (1984); Karlsson et al, Biochem. J. 255:223 (1988)). Endothelial cells secrete both O2xe2x88x92 (Halliwell, Free Radical Res. Commun. 5:315 (1989)) and endothelium-derived relaxing factor, putatively identified as nitric oxide (NOxe2x80xa2) (Noak and Murphy, in Oxidative Stress Oxidants and Antioxidants, eds Sies, H. (Academic, San Diego), pp. 445-489 (1991)). NOxe2x80xa2 functions as a vasoregulator and as a regulator of neurotransmission (Schuman and Madison, Science 254:1503 (1991)). NOxe2x80xa2 can, however, be toxic to neurons in some situations (Dawson et al, Proc. Natl. Acad. Sci. USA 88:6368 (1991)). O2xe2x88x92 is known to inactivate NOxe2x80xa2-induced vasorelaxation (Gryglewski et al, Nature 320:454 (1986); Rxc3xcbanyi and Vanhoutte, Am. J. Physiol. 250:H822 (1986); Rubanyi and Vanhoutte, Am. J. Physiol. 250:H815 (1986); Bult et al, Br. J. Pharmacol. 95:1308 (1988); Nucci et al, Proc. Natl. Acad. Sci. USA 85:2334 (1988)). Thus, a possible function for EC-SOD is to protect NOxe2x80xa2 released from cells from O2xe2x88x92-mediated inactivation.
The reaction of O2xe2x88x92 with NOxe2x80xa2 is also known to produce a potentially toxic intermediate in the form of the peroxynitrite anion (ONOOxe2x88x92) (Beckman et al, Proc. Natl. Acad. Sci. USA 87:1620 (1990); Mulligan et al, Proc. Natl. Acad. Sci. USA 88:6338 (1991); Hogg et al, Biochem. J. 281:419 (1992); Matheis et al, Am. J. Physiol. 262:H616 (1992)). Thus EC-SOD may also function to prevent the formation of ONOOxe2x88x92.
Surprisingly, it has been found that EC-SOD increases, rather than decreases, central nervous system O2 toxicity and that this effect of EC-SOD occurs through modulation of NOxe2x80xa2. This result implicates NOxe2x80xa2 as an important mediator in O2 toxicity. The invention thus relates to methods of manipulating nitric oxide function that involve the use of extracellular antioxidants.
In addition to superoxide radicals, hydrogen peroxide is an oxidant species that is produced under a wide variety of conditions of oxidant stress. The invention thus also provides a method of manipulating hydrogen peroxide levels.
The methods of the invention find application in various disease and non-disease states in which oxidative stress plays a role, including inflammation. In a broader sense, the invention relates generally to methods of modulating intra- and extracellular processes in which an oxidant such as O2xe2x88x92 or hydrogen peroxide is a participant.
The present invention relates to a method of modulating intra- or extracellular levels of oxidants such as superoxide radicals, hydrogen peroxide and peroxynitrite. More particularly, the invention relates to a method of modulating normal or pathological processes involving superoxide radicals, hydrogen peroxide, nitric oxide or peroxynitrite using low molecular weight antioxidants, for example, mimetics of SOD, catalase or peroxidase.
In one embodiment, the present invention relates to an oxidant scavenger, for example, a mimetic of superoxide dismutase, catalase or peroxidase, comprising a nitrogen-containing macrocyclic moiety and a cell surface of extracellular matrix targeting moiety, or a pharmaceutically acceptable salt thereof.
In another embodiment, the present invention relates to oxidant scavenger of the formula: 
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a bond, 
wherein X is a halogen and Y is an alkyl group and wherein 
indicates bonding to R2 at any position and 
indicates bonding to R2 and the substituent at any position; and
R2 is a bond, xe2x80x94(Cxe2x80x22)nxe2x88x92, xe2x80x94(CYxe2x80x22xe2x80x94CYxe2x95x90CYxe2x80x2)nxe2x88x92, xe2x80x94(CYxe2x80x22xe2x80x94CYxe2x80x22xe2x80x94CHxe2x95x90CH)nxe2x88x92, xe2x80x94(CYxe2x80x2xe2x95x90CYxe2x80x2)nxe2x88x92, 
wherein Yxe2x80x2 is hydrogen or an alkyl group and wherein n is 1 to 8; and
R3 is xe2x80x94Yxe2x80x3, xe2x80x94OH, xe2x80x94NH2, xe2x80x94N+(Yxe2x80x3)3, xe2x80x94COOH, xe2x80x94COOxe2x88x92, xe2x80x94SO3H, xe2x80x94SO3xe2x88x92, xe2x80x94Cxe2x80x94PO3H2 or xe2x80x94Cxe2x80x94PO3Hxe2x88x92, wherein Yxe2x80x3 is an alkyl group,
wherein, when R1 is 
and R2 is a bond, R3 is not Yxe2x80x3 (eg xe2x80x94CH3), and
wherein, when R1 is 
and R2 is a bond, R3 is not xe2x80x94Yxe2x80x3 (eg xe2x80x94CH3), xe2x80x94N+(Yxe2x80x3)3 (eg xe2x80x94Nxe2x88x92(CH3)3), or COOH.
In a further embodiment, the present invention relates to an oxidant scavenger of the formula: 
or a pharmaceutically acceptable salt thereof, or metal complex thereof wherein said metal is selected from the group consisting of manganese, copper and iron, wherein:
each R1xe2x80x2 is independently a bond, 
wherein Yxe2x80x3 is an alkyl group, and wherein 
indicates bonding to R2xe2x80x2 at any position and 
indicates bonding to R2xe2x80x2 and the R1xe2x80x2 phenyl substituent at any position;
each R2xe2x80x2 is independently a bond, or xe2x80x94(CH2)nxe2x80x94 wherein n is 1-4,
each R3xe2x80x2 is independently xe2x80x94Yxe2x80x3, xe2x80x94Yxe2x80x2xe2x80x3, xe2x80x94H, xe2x80x94OH, xe2x80x94OYxe2x80x3, xe2x80x94NO2, xe2x80x94CN, xe2x80x94NH2, xe2x80x94COOH, xe2x80x94COYxe2x80x3, xe2x80x94COOxe2x88x92, or a heterocyclic group, wherein Yxe2x80x3 is as defined above and Yxe2x80x2xe2x80x3 is a primary, secondary, tertiary or quaternary amine,
wherein when R1xe2x80x2 is 
R3xe2x80x2 is not COOH, COYxe2x80x3 or COOxe2x88x92,
wherein when R1xe2x80x2 is 
or 
R3xe2x80x2 is not xe2x80x94NO2, and
wherein xe2x80x94R1xe2x80x2xe2x80x94R2xe2x80x2xe2x80x94R3xe2x80x2, collectively, are not xe2x80x94H, 
In yet a further embodiment, the present invention relates to a method of protecting cells from superoxide radical-, hydrogen peroxide- or peroxynitrite-induced toxicity comprising contacting the cells with an oxidant scavenger, eg a SCD, catalase or peroxidase mimetic, sufficient to effect the protection.
In another embodiment, the present invention relates to a method of inhibiting damage due to oxidation of a substance, for example, with the subsequent formation of O2xe2x88x92, hydrogen peroxide, or peroxynitrite comprising contacting the substance with an amount of a SOD mimetic sufficient to effect the inhibition.
In a further embodiment, the present invention relates to a method of inhibiting xanthine oxidase activity of a cell or tissue comprising contacting the cell or tissue with an amount of an oxidant scavenger sufficient to effect the inhibition.
In another embodiment, the present invention relates to a method of treating a pathological condition of a patient (eg, of the lungs of a patient) resulting from superoxide radical-induced degradation of NOxe2x80xa2. The method comprises administering to the patient (eg, to the airways of the patient) an effective amount of a compound having the activity and tissue specificity of SOD (eg EC-SOD) under conditions such that the treatment is effected.
In a further embodiment, the present invention relates to a method of treating in inflammatory condition in a patient in need of such treatment comprising administering to the patient an effective amount of an oxidant scavenger, eg a mimetic of SOD, (eg EC-SOD), catalase or peroxidase, under conditions such that the treatment is effected.
In another embodiment, the present invention relates to a method of treating a disorder resulting from aberrant smooth muscle function in a patient in need of such treatment comprising administering to the patient an effective amount of a mimetic of SOD (eg EC-SOD) under conditions such that the treatment is effected.
In a further embodiment, the present invention relates to a method of modulating physiologic functions of NOxe2x80xa2 in a mammal comprising administering to the mammal an oxidant scavenger in an amount sufficient to effect the modulation. Physiologic functions of NOxe2x80xa2 include its function as a smooth muscle relaxant, neurotransmitter and immune modulator.
In yet a further embodiment, the present invention relates to soluble oxidant scavengers, for example, mimetics of SOD, catalase or peroxidase, and to targeted forms thereof, in particular, mimetics of EC-SOD having a GAG binding moiety attached thereto.
In another embodiment, the present invention relates to an isolated EC-SOD gene sequence, or portion thereof.
Objects and advantages of the present invention will be clear from the description that follows.